Poxviruses include some dangerous emerging or re-emerging pathogens as well as some promising vaccine vectors for infectious diseases and cancers. They are unique among viruses in that they encode a large number of proteins that are dedicated to evading host immune responses. These proteins include secreted inhibitors of cytokines as well as intracellular inhibitors of immune signaling or antiviral factors. The long-term goal of our project is to uncover the mechanisms of poxvirus immune evasion, which will reveal fundamental principles about virus-host interactions and provide knowledge for the development of vaccines and antivirals. The focus of our last funding period was on a secreted poxvirus interleukin-18 (IL-18) binding protein (IL-18BP), which prevents IL-18 from inducing IFN-gamma production. We have been very productive and accomplished all the specific aims. In addition, we discovered in vaccinia virus (VACV) two intracellular inhibitors of type I interferons (IFN), K1 and C71,2. This is an exciting finding, since K1 and C7 are host-range factors essential for poxviruses replication in mammalian hosts but their mechanism of action has long been a mystery. We hypothesize that K1 and C7 are essential for poxvirus replication due to their inhibition of a critical IFN-inducible antiviral pathway. Therefore uncovering the mechanism of action of K1 and C7 will reveal not only molecular basis for poxvirus host tropism but also critical innate immune defense mechanism against poxviruses. In the next funding cycle, we propose to follow up our discoveries on K1 and C7 and pursue the following aims. Aim 1. Structure-function analysis of VACV C7 based on crystal structures of C7 family of proteins. Aim 2. Identify the host antiviral factors that are targeted by K1 and C7. Aim 3. Determine the in vivo role K1 and C7 play in replication capacity and immunogenicity of VACV.